Solid Phase Cell Isolation and/or Enrichment Method

ABSTRACT

The present invention concerns a solid phase method for isolating and/or enriching predetermined cells from a sample. Such a methods are use e.g. to isolate and enrich predetermined cells like fetal cells from a sample of maternal peripheral blood, tumor cells from a sample of body fluid or stem cells from a fluid or fluidized sample of body tissue or body fluid. The solid phase isolation method of the present invention is used for isolating predetermined cells from a sample containing such predetermined cells by binding the predetermined cells to a solid surface. According to the invention the sample is contacted with the solid surface and then removed from the solid surface, wherein the sample or a washing buffer contains a polyol during or after contacting the sample with the solid surface.

The present invention concerns a solid phase method for isolating and/orenriching predetermined cells from a sample. Such methods are used e.g.to isolate and enrich predetermined cells like fetal cells from a sampleof maternal peripheral blood, tumor cells from a sample of body fluid orstem cells from a fluid or fluidized sample of body tissue or bodyfluid.

The detection and analysis of rare cells in blood, bone marrow and otherspecimens becomes an important need in diagnostics: The detection ofcirculating tumor cells, including tumor stem cells and stem cells inepithelial-mesenchymal transition (EMT), and minimal residual cancer isparticularly useful in oncology for improved prognosis, early detectionof disease progression and therapy monitoring. Another application ofsuch techniques is the detection and analysis of fetal cells in maternalblood that enables effective and non-invasive prenatal diagnosis ofgenetic and chromosomal aberrations at early stages of fetaldevelopment.

PCR based DNA analysis or RT-PCR based expression profiling aresensitive and easy to handle technologies for the detection and analysisof such rare cells. However, these technologies are affected by thefact, that contaminating leukocytes as a source of non-specificbackground signals due to inherent illegitimate expression or endogenousexpression of tumor associated antigens are lowering the specificity ofthe detection of rare cells, e.g. circulating tumor cells. Similarly,DNA genotyping is not possible if the isolated tumor or fetal cells arenot sufficiently pure.

The enrichment of cells from bodily specimens is an important task inmany therapeutic applications, too, besides diagnostic applications,since monoclonal antibodies (or other ligands and specifiers) areavailable now that allow the separation of many particular cell typesfrom sources with mixed cell populations such as blood, bone marrow andother tissues. Cell based therapies are a growing field that placesserious demands on the selectivity of any cell isolation technique thatis useful for that purpose. Stem cell graft engineering andimmunotherapy of cancer are one of the most important applications incellular therapy. Autologous stem cells enriched from blood or bonemarrow can be used to support high dose chemotherapy regimens for avariety of malignancies.

There is a great demand for a high purity of cells isolated with cellenrichment technologies:

The usefulness of enriched stem cell products is impaired bycontaminating malignant cells that might be a source for latermetastases and relapses. A contamination with T-cells is the major causeof a graft versus host disease and is the primary reason fortransplantation failure. Thus, an improvement of the efficacy ofexisting therapeutic cell enrichment technologies is a major issue thathas to be urgently addressed.

There are several techniques available for the enrichment of cells fromblood and other specimens. Cell sorting by FACS technology is a veryspecific method that can be applied to enumerate and collect rare cellsfor further use and analysis. However, this method is not applicable yetroutinely to whole blood samples or large volumes and for large scalecell preparations. Several immunochemical methods have been developedfor the enrichment of cells from fluid specimens using solid phaseadsorption as for instance by immunocapturing. Monoclonal antibodies (orother adequate ligands like, for instance, aptamers or other specifiers)can be immobilised on solid surfaces like sepharose, glass, latex orplastic beads or other surfaces for through-flow column or batchapplications. Although the handling of these devices is relativelyconvenient, recovery and purity obtained with such devices isinsufficient in many cases. The use of antibody-labelled magnetic beadsturned out to be quite efficient, showing good recovery rates and anenrichment rate of 4-5 log₁₀. Nevertheless, this purity is still too lowfor expression profiling, especially if the molecular marker of interestis not over-expressed in the target cells as compared to potentiallycontaminating nucleated cells like leukocytes or erythropoietic stemcells. Furthermore, DNA-based genetic typing experiments todifferentiate fetal from maternal cells or tumor from normal cells aregenerally not possible in such samples. Therefore, increasing efficacyof cell purification with solid phase immunochemical or similar devicesis an urgent need not only for diagnostic but also for therapeuticapplications.

There are several reasons for non-specific binding or trapping ofunwanted cells during cell enrichment:

1. Although monoclonal antibodies (or other selected ligands andspecifiers) should exhibit a high specificity towards the antigen (orreceptor) chosen for cell separation, there may still be considerablenon-specific binding to similar antigen or receptor structures.

2. Another reason for trapping unwanted cells may be physicalinteractions (hydrophobic or electrostatic interactions or simplymechanical trapping) with and within the solid phase/antibody (ligand orspecifier) structure.

3. The target antigens or receptors chosen for cell enrichment mightalso be expressed on some non-target cells present in the same specimen(e.g. through illegitimate or endogenous expression).

In order to improve the performance of present days enrichment methods,these methods could be adapted to obtain better specificity by choosinga more specific antibody, ligand or specifier towards a chosen cellsurface target (see 1 and 2 above) whereas non-specific carryover ofnon-target cells cannot be easily prevented (see 3 above). Choosingmagnetic beads as carriers for antibodies, ligands or receptors insteadof through-flow columns filled with beads or choosing polystyrol beadsinstead of sepharose are examples for possible improvements. However,most attempts for technical improvements did not lead to the puritiesrequired in many cases, and there is still an urgent need for a furtherreduction of the amount of non-specifically bound or trapped non-targetcells, preferably by adjusting binding conditions that avoid physicalinteractions.

It is the object of the present invention to provide an improvedisolation and/or enrichment method based on solid phase technology, thatsuccessfully removes or diminishes non-specifically bound or trappednon-target cells during solid-phase capturing like immunocapturing orligand-capturing.

This object is solved by the solid phase isolation and/or enrichmentmethod according to claim 1. Improvements of the inventive method aregiven in claims 2 to 8. Further claims 9 to 14 provide usages of thesolid phase isolation and/or enrichment method according to theinvention.

The present invention enables for the first time the use of solid phaseimmuno- or ligand-capturing procedures for important applications, e.g.genotype expression profiling, that require purified cell populationsobtained from mixed cell populations like blood, bone marrow or similarspecimens. The inventive method successfully removes or diminishesnon-specifically bound or trapped non-target cells during solid phaseisolation like solid phase immunocapturing or solid phaseligand-capturing.

The use of polyols for protein stabilisation is generally known anddescribed. For example, glycerol is frequently used for thecryo-conservation of cells, and other polyols like mannitol areeffectively used for the preservation of red blood cell preparations.Polyols (e.g. sorbitol, sucrose, trehalose) are known to be useful forprotection of bacterial and eukaryotic cells during drying or heat shockprocedures.

Different to these known uses of polyols in the prior art, the inventorsobserved to their great surprise, that polyols may not only be used forpreservation of samples but are also able to reduce the backgroundsignals caused by non-specific bound or trapped non-target cellsdramatically when used in solid phase cell enrichment procedures,whereas at the same time the recovery of target cells was not affected.It can be speculated that interactions of polyols with hydrophobicprotein structures increase the specifity of solid phaseantigen/antibody (ligand or specifier/receptor) interactions possibly bylowering non-specific hydrophobic binding and by stabilizing thesecondary and the tertiary structures of antibodies, ligands orspecifiers and antigens or receptors, thereby enhancing the bindingaffinity and reducing non-specific interactions.

By adding polyols to the sample during solid phase cell enrichmentprocedures either during contacting the sample with the solid phase orin a subsequent washing step of the solid phase, it could be observedthat the amount of non-specifically bound or trapped non-target cellswas reduced by 99%, e.g. when using an inventive 50% (V/V) glycerol/PBSwashing buffer in a manner as described e.g. in the Manual of theAdnaTest BreastCancerSelect (AdnaGen AG, Langenhagen, Germany) usingantibodies against epithelial and tumor-associated antigens (EPCAM,MUC1, HER2) conjugated to magnetic beads. It further turned out that therecovery of target cells was not affected and the concentration of thetarget cells was very low. The addition of polyols, preferably glycerol,to samples before the cell enrichment procedure decreased thenon-specifically bound or trapped non-target cell load substantially.This could also be observed at lower concentrations of glycerol, e.g. 1to 10% (V/V). In the following, some examples of the inventive methodare given.

In these examples,

FIG. 1 shows a schematic overview of the sample preparation andanalysis;

FIG. 2 shows the effect of different glycerol concentrations in theAdnaTest BreastCancerSelect washing buffer on CD45 mRNA levels;

FIG. 3 shows in FIG. 3 a the effect of a washing buffer containing 20%glycerol and 5% mannitol on the recovery of 2 MCF7 tumor cells spikedinto 5 ml blood of healthy donors and FIG. 3 b the decrease of CD45 mRNAlevels due to the modified washing buffer;

FIG. 4 shows the effect of glycerol added to blood samples on thenon-specific binding of leukocytes;

FIG. 5 shows 5 healthy donor blood samples, analyzed in duplicates forestrogen receptor (ER) expression with the AdnaTest BreastCancerDetectfollowed by PCR amplification of ER cDNA developed for this purpose. ERwas amplified in all samples if PBS only was used during the beadwashing step. The average amplicon concentrations were 0.15 ng/μl. Thisnon-specific background activity disappeared when PBS/glycerol was usedfor washing;

FIG. 6 shows the influence of different polyols in the washing buffer onleukocyte background;

FIG. 7 shows the dependency of the purity of CD34(+)-stem cells purifiedthrough anti-CD34 antibody coated magnetic beads on the glycerolconcentration in the washing buffer; and

FIG. 8 shows the effect of polyols on leucocyte background for theisolation of tumor stem cells and EMT cells and subsequent detection ofthe relevant marker expression.

Examples 1-4 have been performed according to the manufacturersinstructions for the detection of CTC.

EXAMPLE 1

5 ml blood samples obtained from healthy donors were processed with theAdnaTest BreastCancerSelect followed by a subsequent determination ofCD45 mRNA expression. After the inoculation of the blood samples withthe magnetic beads of the AdnaTest BreastCancerSelect, the subsequentwashing steps were performed with PBS buffer without or with addition ofdifferent amounts of glycerol (0-50% (V/V)) (see FIG. 2). The CD45 mRNAlevels decrease with increasing glycerol concentrations as shown inFIG. 1. This indicates the disappearance of contaminating leukocytes asthe source of the CD45 mRNA expression.

EXAMPLE 2

2 MCF7 breast cancer cells were spiked into 5 ml blood drawn fromhealthy donors. The tumor cells recovered with the AdnaTestBreastCancerSelect were subsequently analysed for tumor associated mRNAmarkers using the AdnaTest BreastCancerDetect. This was followed by aCD45 PCR to estimate the decrease of the leukocyte contamination. Afterthe inoculation of the blood samples with the AdnaTestBreastCancerSelect magnetic beads, the washing steps were performed withPBS containing 20% (V/V) of glycerol and 5% (W/V) mannitol. As shown inFIG. 3 a, the recovery of the spiked cells was not impaired by themodified washing buffer. Surprisingly, the CD45 and actin mRNAconcentrations decreased at the same time as the amplicon concentrationsof the tumor associated markers increased, indicating a reduction ofcontaminating leukocytes and a higher yield of tumor cell mRNA (FIG. 3b).

EXAMPLE 3

Glycerol was added in different concentrations (0-1% (V/V)) to 5 mlblood samples from healthy donors. The samples were analysed using theAdnaTest BreastCancerSelect/Detect followed by a subsequentdetermination of CD45 mRNA expression. The CD45 amount is decreasingwith increasing glycerol concentrations indicating a lower amount ofbound or trapped leukocytes as shown in FIG. 4.

RT-PCR assay addressing the expression of estrogen receptor (ER) andprogesterone receptor (PR) in circulating tumor cells was developed forinclusion in the AdnaTest BreastCancerSelect/Detect. Since the test forER expression shows a relatively high background due to bound or trappedleukocytes expressing ER, it is unable to surpass a specificity of 80%at the required analytical sensitivity level (i.e. 1 or 2 tumor cells in5 ml blood). This background activity (about 0.15 ng/μl on average),responsible for the reduction of the specificity, can be eliminated ifPBS containing 30% (V/V) glycerol is used in the washing steps as shownin FIG. 5.

EXAMPLE 4

The effect of different polyols in the washing buffer on CD45 expressionin the blood of healthy donors was determined in order to show theability of different polyols to minimize unspecific background.

5 ml blood samples obtained from healthy donors were processed withAdnaTestBreastCancer Select followed by a CD45 RT-PCR. The washing stepswere performed with PBS buffer containing one of said three polyols(sorbitol (10% W/V), fructose (10% W/V), glycerol (10% V/V)). PBS bufferwithout additive was used as a control and for an additional final washin all samples before cell lysis and RT-PCR. Detection of CD45expression is an indicator for selectivity in the separation stepfollowed by detection of CD45 as marker for residual leukocyte cells. Asshown in FIG. 6, all polyols caused a reduction of CD45. Sorbitol andfructose caused about 15% and glycerol about 35% reduction of leukocytebackground. Obviously, as shown with these three arbitrarily selectedpolyols, all polyols are suitable for the present invention.

EXAMPLE 5

In this example, the purity of CD34-positive stem cells from cord bloodafter immunomagnetic enrichment is determined depending on the glycerolconcentration in the washing buffer (see FIG. 7).

Mononuclear cells (MNC), 3.48×10⁸ cells; 9.7×10⁸ cells and 2.59×10⁸cells, obtained from cord blood were re-suspended in 1 ml PBS buffercontaining various concentrations of glycerol (V/V), 5% (W/V) mannitoland 0.1% BSA. After adding magnetic beads (Dynal) with anti-CD34antibodies coupled to them, the suspension was incubated for 30 min inan overhead shaker at room temperature.

After incubation, the beads cell suspension was washed 3 times with 1 mlPBS buffer containing various concentrations of glycerol (V/V) and 5%(W/V) mannitol and 0.1% (W/V) BSA followed by lysis of the bead cellcomplexes in 200 μl lysis buffer (Dynal). After mRNA isolation andreverse transcription, the resulting cDNA was analyzed by PCR for CD45(to determine trapped leukocytes) and CD34 (to determine enriched stemcells) transcripts. A ratio of the quantified PCR signal was calculatedto determine the relative purity of the stem cells in relation to theglycerol concentration in the washing buffer.

As is shown in FIG. 7, glycerol/mannitol containing PBS washing bufferssignificantly increase the purity of the CD34 fraction (stem cellfraction) with increasing glycerol concentration in the washing buffercompared to the washing buffer without any polyol.

EXAMPLE 6

The detection of EMT and tumor cell markers is impeded by the highbackground signals produced by contaminating leukocytes.

To determine the effect of the AdnaWash buffer, containing the polyolsglycerol (23% (V/V)) and mannitol (5% (W/V)) in PBS, healthy donorsamples were processed with the AdnaTest BreastCancerSelect reagentsaccording to instruction with and without addition of this buffer. ThecDNA obtained from these samples was analyzed by PCR for the EMT markersPI3KCA, SIP1 and Akt2 as well as for the tumor stem cell markers ALDH1and BMI1.

As shown in FIG. 8, polyols decrease the leukocyte signals interferingwith EMT markers (Akt2, PI3KCA, SIP1) and tumor stem cell markers (BMI1,ALDH1) analysis due to removal of trapped leukocytes which is confirmedby the decrease of the actin signal.

By this example it is shown that trapped leukocytes express EMT and stemcell markers and produce unacceptable strong background signals. Thesesignals could be efficiently reduced with a polyol containing washingbuffer enabling a specific analysis of these markers on CTC. However,recovery of the CTC was not reduced.

1.-14. (canceled)
 15. A solid phase isolation method for isolatingpredetermined cells from a sample containing the predetermined cellscomprising isolating the predetermined cells by binding thepredetermined cells to a solid surface including contacting thepredetermined cells with the solid surface, and then washing portions ofthe sample which are not bound to the solid surface from the solidsurface with a washing buffer wherein the sample contains a polyol atleast during one of (a) contacting the sample with the solid surface or(b) washing.